What is Real-Time PCR? Its process and applications.

 

The real-time PCR, A game changer after traditional PCR machine. 

A real-time PCR quantify the DNA/RNA in a real time with each cycle it progress. So you don't need to wait for the end, like in traditional PCR. In this blog we will focus on the concept of real-time PCR, its process and its application. 

What is it basically? Just imagine you have only one DNA fragement which has a very unique coding genes which is responsible for a causing particular disease. We need this fragment in a large quantity for the study purpose. Like in a traditional PCR we have to wait for last part of the PCR cycle to happen and then we will go for visualization but in that also we are not getting the exact copy number that we had apmlified. 

To solve this problem Russel Higuchi and his team in 1992 developed real-time PCR. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Using sequence-specific primers, the number of copies of a particular DNA or RNA sequence can be determined. By measuring the amount of amplified product at each stage during the PCR cycle, quantification is possible.

Real-time PCR steps: 

• Denaturation : High temperature incubation is used to “melt” double-stranded DNA into single strands and loosen secondary structure in single-stranded DNA. The highest temperature that the DNA polymerase can withstand is typically used (usually 95°C). The denaturation time can be increased if template GC content is high.

• Annealing : During annealing, complementary sequences have an opportunity to hybridize, so an appropriate temperature is used that is based on the calculated melting temperature (Tm ) of the primers (5°C below the Tm of the primer).

• Extension : At 70-72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in real-time PCR is small, this step is often combined with the annealing step using 60°C as the temperature.

Components are required to setup a real-time PCR are:

• Template DNA/RNA
• Primers
• Taq DNA polymerase
• dNTP
• NFW
• Buffer
• fluoroscent dye (SYBR green) or probe (Taqman)

Types of real-time PCR are: Real-time PCR uses fluorocent dyes or probe to detect and quantify PCR products.

1. SYBR green based
2. Probe based 

Applications of real-time PCR:

• To detect infectious diseases
• Gene expression analysis
• Diseases diagnosis
• Viral quantification
• Mutation Detection
• Cancer research
• GMO detection
• Environmental monitoring

Conclusion: 

Real-time PCR is used to quantify the target seqence in realtime by using the denaturation, annealing and extention of DNA followed by measuring the fluroscence of SYBR green or Taqman probe.